Nieto-Fabregat, Ferran (2022) Screening of microbial glycoconjugates eukaryotic interactors for an effective comprehension of dialogue at mucosal level. [Tesi di dottorato]

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Abstract

This thesis is focused on the understanding at a molecular level of the recognition events at the human microbiota interface. In particular, I focused on the isolation and characterization of bacterial LPS, the major component of Gram-negative outer membrane external leaflet. Glycans dominate the interface between the host (human) and bacteria. Thus, the understanding of most of the biological processes that take place at host-microbiota interface requires the knowledge of structure and function of surface glycans. For this reason, it is pivotal to perform a detailed mapping of bacterial carbohydrates for individual microbiota members as well as analysing their interaction with host immune proteins like lectins as they occur ubiquitously in nature and play key roles in cell-cell communications and immune homeostasis maintenance through recognition of carbohydrates. Therefore, the molecular basis of the interaction between LPS and host immune lectins will be analysed by combination of wet lab, NMR, other biophysical techniques (SPR, ITC, Fluorescence, …) and computational studies.The work has been structured into several chapters summarized below: DC-SIGN: Due to its dual role in mediating both tolerance and immune response, DC-SIGN raised great interest among the scientific community. The modulation of the immune response either in a pro- or an anti-inflammatory response have been exploited by pathogens and tumour cells. In this work has been proved that DC-SIGN has the ability to recognize ligands lacking Man and Fuc residues, by performing interaction studies with a pathogenic bacterium like E. coli and a commensal bacterium like B. vulgatus. Galectins: Galectin-1 and Galectin-3 are among the best-studied galectins mainly due to their involvement in cancer progression. Therefore, the molecular basis of their interaction with inhibitors is required to develop new molecules for therapeutic intervention. Here, two selenoglycosides have been studied by with the aim to shed light towards a better understanding of the interaction basis for the design of specific inhibitors. Siglec-7: Extraction and purification of three different strains of Fusobacterium nucleatum to be further studied with Siglec-7 as they have the ability to present sialic acid or sialic acid like residues in their O-antigen in order to exploit Siglecs inhibition and escape host immune response. Structural models of the interaction between Siglec-7 and two different strains, one containing sialic acid and the other lacking this residue have been proposed. The obtained information not only allowed to prove the ability of Siglecs to recognize glycans lacking sialic acid but also can be valuable in the development of therapies effective against CRC. Siglec-2: Investigation of two sialo-derivatives with the aim to identify novel ligands of Siglec-2 with therapeutic and diagnostic potential. The obtained results give information about the effects of the rigid galactoside moiety in the binding specificity of CD22, contributing to rational design of CD22 potent inhibitors and/or regulators. Mumps virus hemagglutinin neuraminidase: Natural α-2,3-linked sialic acid containing ligand and a synthetic potential inhibitor have been tested in the interplay with mumps virus hemagglutinin neuraminidase (MuV-HN) of strain SBL-1. The structural basis of the interaction and the reaction mechanism were studied to acquire knowledge for the development of novel anti-viral drugs.

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